Replicative gaps in DNA damage tolerance, genome instability, and cancer therapy

Replicative single-stranded DNA gaps are emerging as central intermediates in the cellular response to replication stress. Replication frequently continues past lesions or difficult-to-replicate regions through leading-strand repriming or delayed Okazaki fragment (OKF) maturation, generating structured gaps requiring stabilization and repair. Here, we describe the major routes of gap formation, including polymerase-helicase uncoupling, impaired OKF processing, PrimPol-mediated lesion bypass, and endogenous abasic site accumulation from base excision repair and DNA methylation turnover. We examine the mechanisms that suppress, protect, and resolve these gaps, highlighting RAD51/BRCA2-mediated stabilization, PCNA modifications, PARP1- and CTC1-STN1-TEN1 (CST)-dependent fill-in pathways, and the balance between translesion synthesis and template switching. Finally, we discuss how persistent gaps drive fork degradation, genome instability, and innate immune activation, contributing to explaining the therapeutic vulnerabilities and resistance of cancer cells to PARP, polymerase Q (Pol θ), and ATR inhibitors. This perspective presents a unified model in which timely replicative gap recognition and resolution ensure genome stability.